Fundam. appl. NemalOl., 1992, 15 (6), 563-573
Intraspecific rDNA Restriction Fragment
Length Polymorphism
in the Xiphinema americanum group
Thierry C.
VRAJN,
David A.
WAKARCHUK,
André C.
LÉVESQUE,
and Richard I.
HAMILTON
Agriculture Canada Research Slation, 6660 NW Marine Drive, Vancouver, BC, Canada V6T IX2.
Accepted for publication 29 April 1992.
Summary - Xlphimma americanum sensu law is a plant parasitic nematode vector of nepoviruses, which is widely distributed in
North America. Considerable variation in the capabiliry to vector nepoviruses and in many morphological characters is found
between different populations. The concept of this species has been considerably restricted by sorne authors while others recognized
the variabiliry but hesitated to split the species. In this study we separated populations of the X. americanum group using restriction
fragment length differences in the 5.8s gene and the internaI transcribed spacers (lTS) of ribosomal DNA. Two plasmid clones from
Xiphinema bricolensis (Xb) 18s and 26s ribosomal genes were isolated from a genomic library using a complete repeat of the
ribosomaJ cistron of Caenorhabdilis elegans (Ce) as a probe. Conserved sequences between Xb and Ce were identified and two
21-mer oligonucleotides primers were designed to amplify the ITS region using the polymerase chain reaction (PCR). The 1.5 Kb
amplified product from the ITS region of each of sixteen populations of the X. americanum group was analyzed for restriction length
polymorphisms (RFLP's). The RFPL's were recorded, dissimilariry coefficients were calculated, and a cluster analysis was
generated arranging the sixteen populations as a dendrogram with five clusters. Two populations of X. rivesi were well separated
from other X. americanum populations. X. bricolensis and two populations from Washington State were grouped together, while
X. pacificum and an undescribed population from California were in another cluster. Mixed populations of X. n'vesi and X. americanum from Pennsylvania and West Virginia could not be resolved. The taxonomie separation of this complex of species is an
important step towards determining the vectors of nepoviruses in North America.
Résumé - Polymorphisme intraspécifique de fragments de restrù:tion de l'ADN ribosomal dans le groupe Xiphinema
americanum - Xiphimma amen'canum sensu lato, parasite de plantes et vecteur de nepovirus est présent dans nombre de régions
d'Amérique du Nord. Il existe dans les différentes populations une variabilité importante chez de nombreux caractères morphologiques. La définition de cette espèce a été restreinte d'une manière considérable par certains auteurs tandis que d'autres, tout en
reconnaissant la variabilité, ont hésité à diviser l'espèce. Cette étude sépare cettaines populations du groupe X. americanum en
utilisant la différence de longueur des fragments de restriction de séquences du gène 5.8s et des régions intergéniques transcrites de
l'ADN ribosomaJ. Deux plasmides contenant des fragments clones des gènes ribosomaux 18s et 26s de Xlphinema bricolensis ont été
isolés à partir d'une librairie génomique en utilisant comme sonde l'opéron complet de l'ADN ribosomal de Caenorhabdilis elegans
(Ce). Les séquences homologues entre Xb et Ce ont été utilisées comme amorces d'amplification des ITS utilisant la réaction de
polymérase en chaîne. Le produit d'amplification de chacune des seize populations du groupe X. americanum, un segment d'environ
1.5 Kb, a été analysé pour la longueur des fragments de restriction (RFLP's). Les polymorphismes ont été notés, des coefficients de
dissimilarité entre populations ont été calculés, et une analyse en grappes a permis de grouper les seize populations en cinq groupes.
Deux populations de X. rivesi sont bien séparées des autres populations du groupe X. americanum. X. bricolensis et deux populations
de l'état de Washington sont regroupées, tandis que X. panfieum et une population de Californie non encore identifiée forment une
autre branche. Des populations de Pennsylvanie et de Virginie de l'Ouest, contenant un mélange de X. americanum et de X. n'vesi,
n'ont pu être séparées. L'étude taxinomique de ce complexe d'espèces est un pas important vers la détermination des vecteurs de
nepovirus d'Amérique du Nord.
Key-words: Restriction fragment length polymorphism, ribosomal DNA, Xiphimma americanum group.
Xiphinema americanum Cobb. 1913 is widely distributed in numerous areas of North America. lt is the
vector of severa! economically important nepoviruses :
tobacco ringspot virus (Hibben & Walker, 1971), tomato ringspot virus (Breece & Hart, 1959), peach rosette
mosaic virus (Klos el al., 1967), and cherry rasp leaf
virus (Nyland el al., 1969). The efficiency of transmission of nepoviruses seems to vary between populations
155N 1164-5571/92/06/563 Il
(Georgi, 1998b; Griesbach & Maggenti, 1989). It would
be useful to relate morphological differentiation to virus
vector characters (Tarjan, 1969; Lima, 1975; Luc &
Southey, 1980; Heyns, 1983; Ma!ik & Jairajpuri, 1983;
Lamberti & Golden, 1986; Griesbach & Maggenti,
1990; Cho & Robbins, 1991). Unfortunately, it has not
been possible to establish links between virus vector ability and morphological differences (Griesbach & Mag-
$ 3 / © Galllhier-Viriars - OR5TO/Vl
563
T. C. Vrain et al.
genti, 1989). Fifteen new species were named by Lamberti and Bleve-Zacheo (1979) from their examination
of several hundred populations formerly identified as
X. americanum. The usefulness or the validity of these
relationships have recently been questioned (McKenry,
1987; Georgi, 1998a; Griesbach & Maggenti, 1990).
Thirteen years after their definition, the taxonomic status of many species within the X. americanum group is
still debated.
These nematodes are notorious for their susceptibility to changes in their environment, and have frustrated
many nematologists in their attempts to maintain populations in laboratory or greenhouse culture. This problem has made it difficult to study their biology and to use
ecological or physiological data to separate populations
into different species. Males are extremely rare or nonexistent in the populations examined, suggesting that
parthenogenesis is a common means of reproduction.
Therefore, the taxonomy of this group has been primarily based on morphometrics. However, it is now recognized that many characters used to separate the recently
described species often overlap, making diagnostics at
best difficult and too often merely tentative (Heyns,
1983; Georgi, 1998a; Griesbach & Maggenti, 1990).
Platzer (1981) anticipated that techniques of molecular biology would be applied to help resolve problems
of identification of plant parasitic nematodes, and the
topic has recently been reviewed (Burrows, 1990; Hyman & Powers, 1991). Restriction fragment length poIymorphisms (RFLP's) have been used to separate species and genera of plant parasitic nematodes (Powers &
Sandall, 1988). Comparative analysis of ribosomal
Rl'\J"A and DNA (rDNA) sequences has become a popular tool for the construction of phylogenetic trees and
in the determination of evolutionary relationships. Qu el
al. (1986) used sequences from 28s ribosomal RNA to
demonstrate phylogeny in Helminths. Williams el al.
(1985), separated geographic variants of Drosophila on
the basis of variation in the nontranscribed spacer of
rDNA. While these and other direct analytical methods
are of proven utility, similar analysis with Xiphinema
populations were precluded due to difficulties in obtaining sufficient quantities of nucleic acids.
A complete repeat of the ribosomal cistron of Caenorhabdùis elegans (Ce) has been cloned and sequenced
(Ellis et al., 1986). Like the ribosomal cistron of many
other organisms it contains three spacers separating the
5.8s, 18s, and 26s genes. In this study we have made use
of the polymerase chain reaction (PCR) to amplify a
region of the ribosomal gene (rDNA) and developed a
sensitive method to examine sequence variability within
the 5.8s gene and the internal transcribed spacers (ITS)
of rDNA. We have amplified the ITS region of rDNA
from sixteen North American populations of the
X. americanum group. Cluster analysis of the RFLP's
allowed us to group these populations, and our results
564
have the potential to clarify the status of sorne recently
described species.
Materials and Illethods
NEJ'v1ATODE PREPARATION
Nematodes from field populations in Pennsylvania,
West Virginia, and Iowa State in the USA, and from
Quebec, Canada, were extracted, frozen in water when
still alive, and shipped in dry ice by]. Halbrendt,]. Kotcon, D. Norton, and G. Bélair respectively. Samples of
soil from the states of California, Oregon and Washington in the USA were provided by M. McKenry, R. lngham, and F. McElroy respectively. Several populations
(Dobbins, Firestone, and Murido) were only tentatively
identified as X. americanum. The Xiphinema bricolensis
(Xb) and X. pacijïcum populations were from the type
localities of these species in British Columbia (Ebsary et
al., 1989).
Soils containing Xiphinema nematodes were processed by a sieving flotation technique (Graham et al.,
1988). The organic matter residues and the nematodes
caught on the lower sieve (250 fJ..m diam. pores) were
placed on Baermann pans. Nematodes were colleeted
from the pans every other day. Molten 2.5 % low gel
temperature agarose at 40 oC was added to an equal
volume of the nematode suspension, and the mix quickIy poured into a Petri dish. The nematodes moved to the
surface of the gel in 2 hours or more, leaving all soil
debris behind. They were handpicked, placed in 50 fJ..lof
distilled water in a 1.5 ml silanized (treated with dimethyldichlorosilane) microcentrifuge tube and stored
at - 80 oc.
Ali conditions of nucleic acid isolation, digestion, ligation, transformation, and hybridization, were according
to laboratory manuais by Sambrook et al. (1989).
NUCLEIC AC!OS ISOLATION
Frozen pellets of nematodes were ground in liquid
nitrogen. Total nucleic acids were isolated after digestion of the homogenate with Proteinase K, followed by
phenol/chloroform extraction, treatrnent with RNase,
and precipitation with ethanol. Xb DNA for cloning
purposes was further purified on cesium chloride/ethidium bromide gradients. Plasmid DNA was isolated by
the alkaline Iysis method.
CHARACTERlZATlON OF
CLONES
X.
BRiCOLENSfS RlBOSOMAL
Total Xb DNA was digested to completion with
EcoRt. The DNA fragments were ligated into alkaline
phosphatase-treated pUC 13 (Pharmacia, Uppsala,
Sweden). Competent Escherichia coli NM522 or JM 109
cells were transformed, and selected on ampicillin agar
plates with isopropylthio - B - D galactosidase (IPTG),
and 5 - bromo - 4 - chloro - 3 - indolyl - B - D galâctoside (xGal). Transformants were screened by
hybridization to a clone of an entire ribosomal cistron of
Fundam. appl. NemalOl.
IntTaspecific rDNA in Xiphinema americanum
Table 1. Origin, location and identification of the 16 Xiphinema populations examined. The population labels c to r correspond to
lanes on the agarose gel pietures and cliagrams of Figures 2 to 5.
c
d
e
f
g
h
X. americanum, Fin 8, West Virginia, USA (Kotcon).
X. americanum, Dobbins, Woodlands, Washington, USA (transmits TornRSV, McElroy).
X. americanum, Firestone, raspberry, Vancouver, Washington, USA (TomRSV, McElroy).
X.
X.
X.
X.
j X.
k X.
1 X.
m X.
n X.
o X.
americanum, Corval1is, raspberry, Oregon State University, Oregon, USA (TomRSV, Ingham).
americanum, Murido, Murido vineyard, Highway 43, S.E. Edge, California, USA (McKenry).
americanum, Padier, Kearney Vineyard, S.E. Corner, Parlier, California, USA (McKenry).
rivesi, Frelighsburg, Apple orchard, Quebec, Canada (Belair).
rivesi, Idlewood, black walnut grove, Idlewood County Park, Fioyd County, Iowa, USA (Norton).
americanum, Hinds Farm, Corn, Iowa State University, Story County, Iowa, USA (Norton).
rivesi 92 % and X. americanum 8 %, Ken peach, Permsylvania, USA (Halbrendt).
americanum 92 % and X. n'vesi 8 %, Peach, Permsylvania, USA (Halbrendt).
rivesi, Adams Peach, Permsylvania, USA (Halbrendt).
americanum 84 % and X. rivesi 16 %, Adams Grass, Pennsylvania, USA (Halbrendt).
p X. amencanum 72 % and X. rivesi 28 %, Apple, Pennsylvania, USA (Halbrendt).
q X. pacificum, Plutt, Osoyoos, British Columbia, Canada.
r X. brico!ensis, Ste Claire, Peachland, British Columbia, Canada.
18s primer
26s primer
5' TTGATTACGTCCCTGCCCTTT 3'
26s
NT5
IT5
18s
5.8s
3' GGAA TCA TTGCCGCTCACTTT 5'
IT5
26s
b:::d
pXb101
pXb481
~ig. 1. Position of Xiphinema clones pXb101 and pXb481 flanking the Internal Transcribed Spacer (by homology ta the ribosomal
cistron of Caenorhabditis e!egans), with sequences and positions of 18s and 26s primers.
Ce provided by D. L. Baillie, Simon Fraser University,
Burnaby, Canada. Both ends of selected ribosomal
clones were sequenced by the Sanger dideoxy-mediated
chain termination method. The position of the Xb
rDNA clones, relative to the gene map of the Ce ribosomal operon, was ascertained by computer searches of
the alignment of cloned Xb sequences with Ce ribosomal sequences from the EMBL database, using
SEQNCE PC 3.1 software (Delaney Software Ltd.,
Vancouver, Canada).
AMPLIFICATION
Two 21 nucleotide sequences from clones pXb 101
and pXb481, positioned respectively on the 18s and the
26s genes (Fig. 1), were synthesized as primers in an
Vol. 15, no 6 - 1992
Applied Biosystems DNA synthesizer (Biotechnology
Department, University of British Columbia). The
DNA fragment containing the ITS region was amplified
by PCR. The reaction mixture contained 10 mM Tris
pH 8.3, 50 mM KCl, 2 mM MgCl z' 0.1 % gelatin,
1.25 mM each of dATP, dCTP, dGTP, and dTIP,
0.5 f.lM ofboth primers, 1 ta 50 ng of Xiphinema DNA,
2.5 units ofTaq DNA polymerase, and deionized water
ta a volume of 50 f.ll. A negative control without DNA
added, and a positive control with Ce DNA, were included in each PCR experiment. The amplification was
done in 40 cycles in a thermocycler (Ericomp, San Diego, CA, USA). Each cycle consisted of a denaturation
step at 94 oC for 1 min, annealing at 55 oC for 1 min,
and polymerization ar 72 oC for 1 min. Ramping rime
was about 10 min per cycle.
565
T. C. Vrain et al.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
The product of amplification was digested with fourteen restriction enzymes for 6 hours in the recommended buffers. The enzymes used were: Apa l, Alu l,
BamH l, BgI l, Dde l, Hind 1II, HinfI, Mbo l, Msp l,
Pal l, Pst l, Pvu II, Rsa l, and Xba I. The DNA fragments thus generated were separated by electrophoresis
in a 2.50 % low gel temperarure agarose slab in TrisacetatelEDTA (TAE) buffer for 90 min. at 50 V. The
marker DNA was pBR328 cut with Bgll and Hinfl, giving band sizes at 2.2 kb, 1.8 kb, 1.2 kb, 1.0 kb, 650 bp,
520 bp, 450 bp, 390 bp, 300 bp, 225 bp, 150 bp
(Boehringer Mannheim). For each nematode population the bands obtained with six enzymes were recorded
as a string of 0 and 1 corresponding to the absence or
presence of individual bands. Data from ail digests that
gave polymorphisms were used for a total of 50 bands.
A matrix X of 0 and 1 was generated with nematode
populations as rows and bands as columns. A new matrix was generated by multiplying the X matrix by its
transpose X' giving the matrix M with nematode populations as rows and columns. The numbers in the diagonal of the matrix M represent the total number of
bands in a particular nematode population and the other
numbers represent the number of bands shared by each
population pair. Dissimilarity coefficients for ail pair
comparisons were calculated using the matrix M and a
Fortran subroutine (Lévesque el al., 1992). From these
values, a cluster analysis by the unweighterl pair-group
method with average (UPGMA) was performed and a
tree was constructed (SAS, 1985).
Results
The yield of Xiphinema nematode extractions from
soil were rather low, averaging 100 to 200 per L of soil.
The quantity of DNA extracted from Xb nematodes
was variable but averaged 0.5 ng per nematode. The
numbers of nematodes available from the other populations srudied were relatively small and although yield
efficiencies were not taken, they were assumed to be
sirnilar.
Six hundred colonies of transformed cells were picked
from the Xb library. Five clones were shown to contain
Xb rDNA by hybridization with 32p labelled Ce ribosomal repeat. The position of two of these clones was of
particular interest ta this work. Clone pXb 10 1 had 277
base pairs (bp), encompassing the 3' end of the 18s gene
and the 5' end of the ITS (Fig. 1). Clone pXb481 was
approximately 1.3 Kb, encompassing the 3' end of the
ITS and the 5' end of the 26s gene. The two 21 nucleotide sequences utilized for primers were completely homologous between Xb and Ce, and other nematode sequences found in the EMBL database. The 18s primer
sequence was 5' TTGATTACGTCCCTGCCCTTT
3', and was aligned with nuc1eotides 2503 to 2523 of the
Ce ribosomal cistron (EMBL databank, accession num566
ber X03680). The 26s primer sequence was 5'
TTTCACTCGCCGTTACTAAGG 3', and was the
reverse complement of nuc1eotides 3774 to 3794
(Fig. 1).
Amplification of template DNA was usuaily lOs to 10 6
fold. The reaction yielded 2 to 5 f.lg of DNA starting
with total DNA from approximately 100 nematodes.
The amplification of the ITS region of each Xiphinema
population gave one fragment approximately 1.~ Kb
long (Fig. 2). The 1.5 Kb fragment hybridized strongly
with c10ned Xb ITS. There were several instances when
nonspecific products of amplification were obtained,
which did not hybridize to the c10ned Xb ITS region
(data not shown). lt was assumed that variations in the
conditions of the PCR reactions were responsible for the
nonspecific amplification. The amplifications were repeated using " Perfect Match" (Stratagene, California,
USA) which usuaily eliminated the formation of nonspecific products. No PCR product was ever obtained in
the negative control treatment lacking DNA template.
The Ce positive control always gave a strong band at
approximately 1.4 Kb (Fig. 2).
Digestion of the amplification product with six of the
fourteen enzymes gave sharp restricted DNA bands on
the agarose gels. There were no restriction sites for BgI l,
Hind lII' Pst l, Pvu II, and only partial digestions with
Apa l, Pal l, Rsa l, and Xba 1.
The pattern of restriction bands obtained with Alu l,
Barn H l, Dde l, Hinf l, Mbo l, and Msp 1 (Diagrams of
Figs. 3 a 8) allowed the grouping of the sixteen populations into five c1usters (Fig. 6). Cluster 1 contained the
two populations from Washington State and the population of Xb from British Columbia. Cluster 2 contained a population from California and the population
of X. pacificum from British Columbia. Cluster 3 contained two populations of X. rivesi from Iowa and from
Quebec. Cluster 4 contained the " Parlier " population
from California recently described as X. amen'canum
sensu laiD (Griesbach & Maggenti, 1990), a population
from Iowa and one from Oregon. Cluster 5 contained ail
the populations from Pennsylvania and West Virginia
labelled X. americanum, X. rivesi, or mixed populations
of these.
Discussion
In preliminary experiments the purity of the DNA
extracted from nematodes in soil was inadequate for
digestion and c1oning. Ir proved important to have the
nematodes free of any soil organic matter residues. Nematodes extracted in Baermann pans were thus further
cleaned of soil debris by their migration wough agarose.
The sequences of the two prirners were from the conserved 18s and 26s genes. The amplification product
included 79 nucleotides from the 5' end of the 26s gene,
and 170 nucleotides from the 3' of the 18s gene. These
conserved sequences, as weil as the 5.8s gene sequence,
Fundam. appl. NemalOl.
Kb
m n
k
a01cdefgh
0
p q
r
2.2"
1.2 ..
Fig. 2. Amplified Internai Transcribed Spacers region of sixteen Xiphinema spp, Ethidium bromide stained 2,5 % agarose gel. a:
DNA marker, pBr 328!BgI l/Hinf 1; 0 : no DNA, amplification control; 1: CaelUiThabdllls elegans ITS reglon; c : ta r : populations of
Xiphinema as in Table l,
Kb
k
abcdefgh
abc
d
e
f
g
h
i
j
k
l
m
n
m
n
0
p
q
r
0
p
q
r
Fig. 3. Restriction fragments of amplified Internai Transcribed Spacers of Xiphinema spp, Ethidium bromide stained 2,5 % aga rose
gel - Hinf 1 digest - a : DNA marker, pBr 328IBgI IlHinf 1; b: unrestricted ITS X. amencanum (VXI A, USA); c : X. amen'canum
(WV, USA); d : X. americanum (WA, USA); e : X. amen'canum (VXI A, USA); f: X. amerUanum (OR, USA); g : X. americanum (CA,
USA); h : X. amen'canum (CA, USA); i: X. rivesi (PQ, Canada); j : X. rivesi (lA, USA); k : X. americanum (lA, USA)~ 1: X. rivesi
92 % and X. amen'canum 8 % (PA, USA); m: X. americanum 92 % and X. rivesi 8 % (PA, USA); n: X. n'vesi (PA, USA); 0 :
X. americanum 84 % and X. rivesi 16 % (PA, USA); p : X. americanum 72 % and X. rivesi 28 % (PA, USA); q: X. pacificum (BC,
Canada); r: X. bricolensis (BC, Canada),
Vol, 15, nO 6 - 1992
567
T. C. Vrain et al.
Kb
ab
cd
abc
efgh
d
e
f
g
h
k
i
j
k
m
l
m
n
n
p
0
0
p
q
q
r
r
Fig. 4. Restriction fragments of amplified InternaI Transcibed Spacers of Xiphinema spp. Ethidiwn bromide stained 2.5 % agarose
gel- Msp 1 digest - (low case leners : see Fig. 3).
amplified with the ITS region of each population were
represented in sorne of the common bands found in ail
populations (Figs 3 - 8).
The choice of the 5.8s gene and ITS fragment for the
restriction polymorphism analysis of the 16 populations
proved to be judicious. Ribosomal DNA is often represented in many copies in the genome of many organisms, and represents an excellent target for the amplification, since only small amounts of nematodes were
available. A fragment from a conserved region, such as
the coding regions of the 18s or the 26s genes, would be
expected to show few polymorphic restriction sites, and
may not have separated the Xiphinema populations.
Converselya fragment ofDNA picked at random might
have shown too many polymorphic restriction sites ta be
useful in grouping the populations. The region we amplified showed both common and polymorphic restriction sites.
Amplification of the 5.8s gene and of the ITS region
of the ribosomal cistron of Xiphinema had the conve568
nience of visualizing polymorphism directly on agarose
gels. This technique allowed us to work with relatively
smaJl quantities of DNA extracted from few nematodes
and there was no need for Southern transfers and hybridization with a 32P-labelled probe.
Griesbach and Maggenti (1989) established that
X. amen'canum sensu stnào is in California, that X. n'vesi
is distinct from X. americanum, and that X. califomicum
is not separable from X. amencanum. In our srudy, the
X. amen'canum sensu lata "Parlier" population from
California was related to another West Coast population
(from Oregon), and a more centrally located population
(from Iowa), both described as X. amen'canum (Ingham
& Norton, pers. comm.).
As weil, X. n'vesi populations from Quebec and Iowa
were grouped in the same cluster and were distinct from
other populations. The DNA of X. rivesi (lanes i and j,
Fig. 5) lacked a BamH 1 restriction site present in ail
other populations of the X. amen'canum group. Ebsary el
al. (1984) and Wojtacic el al. (1982), utilizing standard
Fundam. appl. NemalOl.
lntraspecijic rDNA in Xiphinema americanum
Kb
abc
d
e
f
9
h
abc
d
e
f
g
h
k
m
n
m
n
0
p
q
r
0.45 ..
0.15 ..
i
j
k
l
0
p
q
r
Fig. 5. Restriction fragments of amplified Internai Transcribed Spacers of Xiphinema spp. EÙ1idium bromide stained 2.5 % agarose
gel - Dde 1 digest - (low case leners : see Fig. 3).
morphometrics, had no difficulty in differentiating the
twO species. Georgi (l988a) confirmed the validity of
the separation of these twO species in New York State,
but found important regional differences, from Eastern
to Western New York, with one population displaying
characteristics of bath species. These twO species were
found mixed in sorne instances in New York (Georgi,
1988a), as they often were in Pennsylvania and West
Virginia (Kotcon & Halbrendt, pers. comm.). In Iowa,
these twO species are found in different habitats: X rivesi in wooded areas, and X americanum in corn fields
and prairies (Norton, pers. comm.).
X pacijü;um) a new species from British Columbia
(Ebsary el al.) 1989), was described as somewhat resembling Xcahfornicum. The RFLP study placed it in
the same cluster as " Murido ", an undescribed population from California resembling X calijornicum
Vol. 15, nO 6 . 1992
(Brown, pers. comm.). The dynamics and pathogenicity of this population are noticeably different from the
" Parlier" population (McKenry, pers. comm.). A detailed morphometric study of the " Murido " population
should confum that it belongs to one or the other species, X pacijicum or X calijornicum. The Xb population from British Columbia in cluster 1 is with 2 populations from adjacent Washington State, sent ta us as
X americanum. However these twO populations were
not exarnined criticaily before the RFLP study, and it is
probable that a detailed characterization will place them
away from X. amen'canum sensu slnào.
Several populations were sent ta us as mixtures of
X americanum and X rivesi (Apple, PA; Adams Grass,
Pa; Ken peach, PA; Peach, PA), while others were
tagged as pure X americanum (Fin 8, WV), or pure
X rivesi (Adams Peach, PA). The stylets of 25 nema569
T. C. Vrain et al.
Kb
abcdefg
h
abc
h
m
k
n
0
p
q
r
p
q
r
1.2~
0.45
~
0.15 ~
d
e
f
g
i
j
k
1
m
n
0
Fig. 6. Restriction fragments of amplified Internai Transcribed Spacers of Xiphinema spp, Erhidium bromide stained 2.5 % agarose
gel - Mbo 1 digest - (low case leners : see Fig. 3).
rodes were measured (Halbrendt & Hill, PA; Kotcon,
WV) for ail the populations. Based on these measurements the populations were defmed as containing varying proportions of X. amen'canum and X. n·vesi. Natmally, mixtures of nematodes would blur the results of
the DNA analysis. Samples containing DNA from t,vo
species should have the same pattern of restricted DNA
bands on the electrophoresis gel, albeit with bands of
varying densities corresponding ro the proportions of
each species. This was easily seen with the BamH 1 digests, where populations of X. n'vesi (Janes i and j; Fig.
8) show no restriction site, and populations of the
X. americanum group (Janes d, f, h, and k; Fig. 8) show
one restriction site. The mixed populations show ail
three bands, with the unrestricted fragment prominent
when X. rivesi is a large proportion in the mixture (Janes
1and n, Fig. 8), or with the unrestricted fragment quite
faint when X. n'ven' is a small proportion (Jane c, m and
p, Fig. 8). It appeared that the pure populations of
570
X. amen'canum (Fin 8) and X. rivesi (Adams Peach)
from West Virginia and Pennsylvania were also certainly
mixed populations, since the RFLP patterns showed
characteritics of both species.
The technique described here measures the discontinuity of variation berween taxa otherwise difficult to
separa te by standard morphomeu"ics. In this rDNA
analysis, X. americanum, " Padier" population, recognized as X. amen'canum sensu smào by Griesbach and
Maggenti (1990), was in a separate cluster from X. n'ves!.
It appears that this molecular approach is capable of
separating species within the X. americanum group, and
therefore we conf1rm the validity of X. bricolensis and
X. paclficum, which were in other clusters, well separated from X. americanum sensu stricto or X. rivesi. This
RFLP analysis will help taxonomists decide how much
genetic variability is expressed, and which morphometric characters are useful for identification.
Fundam. appl. NemalOl.
Intraspecific TDNA in Xiphinema americanum
Kb
abcde
fgh
k
m n
0
p
q
r
m
0
p
q
r
0,45 ~
O,15~
abc
d
e
f
g
h
i
j
k
l
n
Fig. 7. Restriction fragments of amplified Internal Transcribed Spacers of Xiphinema spp. Ethidium bromide stained 2.5 % agarose
gel- Alu 1 digest - (low case letters : see Fig. 3).
References
BREECE, J. R. & HART, W. H. (1959). A possible association
of nematodes with the spread of peach yellow bud mosaic
virus. Pl. Dis. RepiT, 43 : 989-990.
BURRows, P. R. (1990). The use of DNA to identify plant
parasitic nematodes. Helminth. AbsIT., 59: 1-8.
CHO, M. R. & ROBBINS, R. T. (1991). Morphological variation among 23 Xiphinema americanum populations. J. Nemawl., 23 : 134-144.
EBSARY, B. A., POlTIER, J. W., & ALLEN, W. R. (1984). Redescription and distribution of Xiphinema rivesi Dalmasso,
1969 and Xiphinema americanum Cobb, 1913 in Canada
with a description of Xiphinema occiduum n. sp. (Nematoda :
Longidoridae). Canad. J. Zoo!.) 62: 1696-1702.
EBSARY, B. A., VRAIN, T. c.) & GRAHAM, M. B. (1989). Two
Vol. 15, nO 6 - 1992
new species of Xiphinema (Nematoda : Longidoridae) from
British Columbia vineyards. Canad. J. Zool.) 67: 801-804.
ELLIS, R. E. SULSTON, J. E., & COULSON, A. R. (1986). The
rDNA of C. elegans : sequence and structure. Nucleic Acids
Res.) 14: 2345-2364.
GEORGI, L. (l988a). Morphological variation in Xiphinema
spp. from New York orchards. J. Nematol.) 20: 47-57.
GEORGI, L. (l988b). Transmission of Tomato Ringspot virus
by Xiphinema americanum and X. rivesi from New York appIe orchards. J. Nematol., 20 : 304-308.
GRAHAM, M. B., EBSARY, B., VRAIN, T. C., & WEBSTER,
J. M. (1988). Distribution of Xiphinema bricolensis and Xiphinema pacijicum in vineyards of the Okanagan and Similkameen valleys, British Columbia. Canad. J. Pl. Palh., 10 :
259-262.
571
T. C. Vrain et al.
Kb
abc
d
e
f
9 h
abc
d
e
f
g
m
k
n
p q
0
r
1.2 ..
0.45 ..
h
i
j
k
l
m
n
0
p
q
r
Fig. 8. Restriction fragments of amplified Internal Transcribed Spacers of Xiphinema spp. Ethidium bromide stained 2.5 % agarose
gel - BamH 1 digest - (Iow case leners : see Fig. 3).
GRIESBACH,J. A., & MAGGENTI, R. (1989). Vector capability
of Xiphinema americanum sensu talO in California. J. Nema1O!., 21: 517-523.
GRIESBACH, J. A., & MAGGENTI, R. (1990). The morphometrics of Xiphinema americanum sensu LalO in California.
Revue NémalOl., 13: 93-103.
fuYNs, J. (1983). Problems of species delimitation in the genus Xiphinema, with special reference to monosexual species, ln: Stone, A. R., Plan, H. M. & Khalil, L. F. (Eds).
ConceplS in NemalOde syslemalics, London, Academie Press:
163-174.
HIBBEN, C. R., & WALKER,J. T. (1971). Nematode transmission of the ash strain of tobacco ringspot virus. Pl. Dis. Replr,
55: 475-478.
HYMAN, 8., & POWERS, T. (1991). Integration of classical and
molecular approaches in nematode raxonomy. A. Rev. PhyIOpalh., 29: 89-107.
KLos, E. ]., FRONEK, K., KNŒRIM, J. A., & CATION, D.
(1967). Peach rosene mosaic transmission and control srudies. Quar€. Bull. Michigan St. Univ. agric. EX!. SLaLn, 49 :
287-293.
572
LAJVlBERTI, F., & BLEVE-ZACHEO, T. (1979). Sructies on Xiphinema americanum sensu talO with description of 15 new
species (Nematoda, Longidoridae). NemalOl. medit., 7 : 51106.
LAJVlBERTI, F., & GOLDEN, A. M. (1986). On the identity of
Xiphinema americanum sensu talO in the nematode coUection
of Gerald Thome. Nemawlogia medit, 14 : 163-171.
LEVESQUE, C. A., BECKENBACH, K., BAILLŒ, D. L., & RAHE,
J. E. (1992). Pathogenicity and differentiation by DNA restriction fragment length polymorphisms of isolates of rwo
Pylhium spp. that colonize Glyphosate-treated plants. Mycologia (in press).
LIMA, M. B. (1965). SLUdies on species of lhe genus Xiphinema
and omer nemawdes. PhD thesis, University of London, England. 165 p.
Luc, M., & SOl.JTHEY, J. F. (1980). Srudy of biometrical variability in Xiphinema insigne Loos, 1949 and X. elongaLUm
Schuurmans Stekhoven & Teunissen, 1938, description of
X. savanicota n. sp. (Nematoda: Longidoridae), and commenrs on thelytokous species. Revue Némawl., 3 : 243-269.
MALU<, Z., & lAIRJ\IPURl, M. S. (1983). Sratistical analysis of
Fundam. appl. Nemawl.
Intraspecijic rDNA in Xiphinema americanum
e: X. americanum (?l, Dobblns (WA, USA)
1
r: X. bricolensis, Ste Claire (BC, Canada)
d: X. americanum (?l, Firestone (WA USA)
q: X. pacificum, Plutt (BC, Canada)
2
1
1
g: X. americanum (?l, Murldo (CA, USA)
-
J:
3
X. rivesi, ld1ewood (lA USA)
1
i: X. rivesi,
1
'-
~
Frellghsburg (PQ, Canada)
k: X. americanum, Hlnds Farm (lA USA)
ri
h: X. americanum, Parller (CA USA)
f: X. americanum, Corvallis (OR USA)
'--
n: X. rivesi, Adams Peach (PA USA)
1: X. rivesi 92%. X. americanum 8%. Ken Peach (PA USA)
5
0: X. americanum 84%, X. rivesi 16%, Adams Grass (PA, USA)
1
1
'----
m: X. americanum 92%, X. rivesi 8% , Peach (PA USA)
p: X. americanum 72%, X. rivesi 28%, Apple (PA USA)
c: X. americanum, Fln 8
rwv,
USA)
Fig. 9. ReJationships of sixteen populations of the Xiphinema americanum group. Dendrogram constructed from dissimiJariry
coefficients, obtained from presence and position of restriction fragments of amplified Internai Transcribed Spacers region. (The
diagnosis of populations Dobbins, Firestone, and Murido is probably inaccurate.) (Iow case leners : see Fig. 3).
variabiliry in Xiphinema americanum Cobb, 1913 sensu Siddiqi, 1959. Indian J. NemalOl., 13 : 71-78.
MCKENRY, M. (1987). Lener to the editor. Pl. Dis., 70: 864.
NYLAND, G. LOWNSBERY, B. F., LOWE, S. K., & MITCHELL,
]. F. (1969). The transmission of cherry rasp leaf virus by
Xiphinema americanum. Phylopalhowgy, 59: 1111-1112.
PLATZER, E. G. (1981). Potential use of protein panerns and
DNA nucleotide sequences in nematode taxonomy. In:
Zuckerman, B. E., & Rhode, R. E. (Eds.) Plant parasiLic
nemal.Odes, Vol. Ill, London, New York, Academie Press:
3-21.
POWERS, T. O., & SANDALL, L.]. (1988). Estimation of genetic divergence in Mewidogyne mt DNA. J. NemalOl., 20 :
505-511.
Qu, L. H., HARDMANN, N., GIU.., L. CHAPPELL, L., NICOLOSO, M., & BACHELLERIE,]. P. (1986). Phylogeny of Helminths determined by rRNA sequence comparison. Molec.
bioch. Parasil.Ol., 20 : 93-99.
Vol. 15,n o 6-1992
SAMBROOK, J., FRlTSCH, E. F. & MANIATIS, T. (1989). Moleeular cwning: a laboralory manual. Cold Spring Harbor
Laboratory Press, 1590 p.
SAS INSTlTUTE INC. (1985). SAS User's guide: Slalislics, Version 5. Cary, NC, USA, 956 p.
TAR,lAN, A. C. (1969). Variation within the Xiphinema americanum group (Nematoda: Longidoridae). NemalOwgica,
15: 241-252.
WILLIAMS, S. M., DE SALLE, R., & STROBECK, C. (1985).
Homogenization of geographical variants at the nontranscribed spacer of rDNA in Drosophila mercalorum. Molec.
Biol. Evol., 2 : 338-346.
WOjTOWlCZ, M. R., GOLDEN, A. M., FORER, L. B.,
& STOUFFER, R. F. (1982). Morphological comparisons between Xiphinema rivesi Dalmasso and X. americanum Cobb
populations from the eastern United States. J. NemalOl., 14 :
511-516.
573